Lesbian, gay, and bisexual populations face barriers accessing health care in Chicago, Illinois. To describe the prevalence of up-to-date cervical cancer screening among lesbian, gay, and bisexual vs heterosexual cisgender women in Chicago. This retrospective, cross-sectional, population-based study of cisgender women residing in Chicago was completed from 2020 to 2022 using data from the Healthy Chicago Survey, which is conducted annually by the Chicago Department of Public Health. Participants included cisgender women aged 25 to 64 years with no history of hysterectomy. Respondents who self-identified as lesbian, gay, or bisexual or other than straight, lesbian, or bisexual were coded as lesbian, gay, or bisexual (LGB). Respondents who self-identified as straight were coded as heterosexual. Those who reported having a Papanicolaou test within the past 3 years were considered up-to-date with cervical cancer screening. Data analysis was performed from June to October 2023. The primary exposure was sexual orientation. Covariates included age, income level, race, ethnicity, having a primary care practitioner (PCP), and insurance coverage. Prevalence ratios (PRs), log-based regression models, and interaction analysis were used to describe the association of sexual orientation with up-to-date screening. The sample included 5167 cisgender women (447 LGB and 4720 heterosexual), aged 25 to 64 years, with no history of hysterectomy. Among LGB cisgender women, 318 (71.14%) reported previous cervical cancer screening compared with 3632 (76.95%) heterosexual cisgender women. The prevalence of up-to-date screening was 10% lower in the LGB group compared with the heterosexual group (PR, 0.90; 95% CI, 0.82-1.00). In regression analysis, having a PCP (PR, 1.43; 95% CI, 1.29-1.59) was associated with up-to-date screening. In interaction analysis, LGB cisgender women with a PCP were 93% more likely to be up-to-date compared with those without a PCP (PR, 1.93; 95% CI, 1.37-2.72). In this cross-sectional study of cervical cancer screening rates between the heterosexual and LGB populations in Chicago, up-to-date cervical cancer screening was associated with having a PCP, regardless of sexual orientation, but this association was greater for LGB individuals. Although LGB populations were less likely to be screened, this disparity may be reduced with more consistent health care access and established care with PCPs.